Acidified Citrate Cysteine Medium (ACCM) that supports approximately 3 logs of Coxiella growth under microaerobic (2.5% O2) conditions. We have now developed a serum-free version of ACCM termed ACCM-2 that supports improved growth of both virulent and avirulent strains of Coxiella in both liquid and solid media. For example, we now routinely obtain >4 logs of growth in liquid media. Moreover, we have increased the colony size on agarose plates by roughly 5-fold to approximately 0.5 mm, greatly aiding clonal isolation by colony picking. Coxiella was successfully cultured in ACCM-2 when medium was inoculated with as little as 10 genome equivalents contained in tissue homogenates from infected SCID mice, providing a sensitive means of primary isolation of the pathogen from Q fever patients. A completely axenic Coxiella genetic transformation system was developed using ACCM-2 that allowed isolation of transformants in about 2 weeks. Transformation using RF1010 ori-based shuttle demonstrated a transformation frequency of approximately 5 x 10-5. Moreover, both CAT and Kan genes are excellent selectable markers for Coxiella transformants in both ACCM as well as cell culture. Both antibiotics are Select Agent compliant, allowing two transformation procedures in a single strain. ACCM-2 will facilitate biochemical studies and, as described below, development of Coxiella genetic systems. Coxiella plates with high efficiency on ACCM-2 agarose ( 1 to 2 genome equivalents per colony) and the resulting colonies contain clonal populations. Colonies can be expanded by direct transfer into ACCM-2 and resultant liquid cultures stably archived. 2) Development of new tools for Coxiella genetic manipulation ACCM culture techniques were used to develop an optimized Himar1-based transposon (Tn) mutagenesis system for Coxiella. In our original Himar1 system, the promoter from CBU1169 (1169P), which encodes the small heat shock protein Hsp20, was used to drive expression of CAT and mCherry red fluorescent protein genes as a single transcriptional unit. Expression was sufficient to confer resistance to 5 &#956;g/ml of chloramphenicol, but mCherry fluorescence was low. Therefore, a new Himar1 transposon plasmid (pITR-CAT:: 311P-MC) was constructed that employs the CBU0311 (outer membrane porin P1) promoter (311P) to drive expression of the mCherry red fluorescent protein gene independent of 1169P-driven CAT. CBU0311 is constitutively expressed at an approximately 6-fold higher level than CBU1169. By both fluorescence microscopy and flow cytometry, transformants harboring this Tn show significantly enhanced mCherry red protein fluorescence and are useful in pathogen-host cell interaction studies. With the optimized Himar1 Tn, we are now constructed ordered libraries of random Coxiella Tn mutants for wholesale testing of individual mutant clones for defects in macrophage invasion and/or replication. An important recent discovery aided by ACCM culture is that vectors derived from the IncQ plasmid RSF1010 autonomously replicate in Coxiella. Shuttle vectors containing the RSF1010 ori are compatible with the endogenous Coxiella plasmid and have a copy number of roughly 3 to 6. This discovery allowed construction of reporter plasmids for identification of proteins secreted by Coxiella into the host cell cytosol. The secretion assay detects cytosolic delivery of &#946;-lactamase (BlaM) or camodulin-activated andenylate cyclase (CyaA) C-terminally fused in-frame to a Coxiella protein containing a translocation signal. In pJB-CAT-BlaM, blaM and CAT are driven independently by constitutive 1169P. Use of each vector verified cytoplasmic translocation of a unique set of 6 suspected C. burnetii secreted proteins. We have modified the backbone of pJB-CAT-BlaM (i. e., pJB-CAT) to contain a cloning site that allows 1169P-directed synthesis of proteins with N- or C-terminal epitope tags of 2xHA or 3xFLAG. A version encoding kanamycin instead of chloramphenicol resistance (pJB-KAN) has also been constructed. Applications of these vectors include expression of dominant/negative proteins for functional studies, expression of epitope tagged proteins to assess protein binding partners and intracellular trafficking, and in trans complementation of mutants generated by Tn or other mutagenesis techniques to fulfill molecular Kochs postulates. The establishment of pJB-CAT/KAN and other RSF1010 ori-based based shuttle vectors is a significant advance in Coxiella genetic manipulation. However, a potential downside of their use is unwanted gene dosage effects due to multiple plasmid copies. Therefore, we developed a system for site-specific single-copy Coxiella gene knock-ins using Tn7. The Tn7 transposase, encoded by tnsABCD, recognizes a specific 30 bp site termed attTn7 that, in Pseudomonas aeruginosa and other gram-negative bacteria, is located in the extreme 3 end of glmS encoding glucosamine-6-phophosphate synthetase. Transposition is orientation specific and occurs 36 bp downstream of attTn7 in an intergenic region. Analysis of the 3 end C. burnetii glmS (CBU1787) revealed a putative attTn7 site. To test whether Tn7 can utilize this site, a two-suicide plasmid Tn7 system was constructed. The plasmid pTNS2::1169P-tnsABCD carries the transposase genes tnsABCD under control of 1169P. The plasmid pMiniTn7T-CAT::MC carries Tn7 with CAT and mCherry red fluorescent protein genes driven independently by 1169P and 311P, respectively. Co-electroporation with both plasmids and selection of chloramphenicol resistant Coxiella in ACCM yielded transformants with Tn7 inserted 36 bp downstream of the predicted attTn7 site in an intergenic region between glmS (CBU1787) and CBU1788, which encodes a hypothetical protein. The transformant exhibits bright mCherry red protein fluorescence in infected Vero cells at 5 days post infection with no obvious growth defect relative to wild-type bacteria. Tn7 was used in extensively in complementation studies of a Coxiella mutant containing a Himar1 insertion in icmD (Project AI000931-09). The mutant exhibits deffective type IV secretion of effector proteins and fails to replicate in macrophages. We also developed a Tn7-based system of anhydrotetracycline inducible gene expression to evaluate the temporal requirements of type IV secretion in Coxiella infection. Stringent control of gene expression by Coxiella growing in ACCM or macrophage host cells was achieved by using anhydrotetracycline induction of the TetRA repressor-promoter fragment. 3) Re-sequencing microarrays reveal genetic polymorphisms of clonal phase II isolates likely responsible for LPS phase variation. The high passage phase II isolates in our stock collection are not clonal and contain a small subpopulation of Coxiella still expressing full-length phase I LPS. The resulting mixed genotype complicates identification of indels (insertions/deletions) strictly associated with phase variation. To circumvent this problem, we used our micromanipulation cloning procedure to isolate clonal phase II populations of high passage Nine Mile, Australia and California isolates. By hybridizing their genomic DNA to a high-density microarray that contains probe sets encompassing the entire genome of the Nine Mile phase I isolate, we identified common indels in phase II organisms that may account for defective LPS biosynthesis.